Available online at www.sciencedirect.com

Nuclear Medicine and Biology 36 (2009) 419 – 426
www.elsevier.com/locate/nucmedbio

Radio-copper-labeled Cu-ATSM: an indicator of quiescent but clonogenic
cells under mild hypoxia in a Lewis lung carcinoma model
Myungmi Oha , Takeshi Tanakaa , Masato Kobayashib , Takako Furukawac , Tetsuya Morid ,Takashi Kudob , Shigeharu Fujiedaa , Yasuhisa Fujibayashib,⁎aDepartment of Otorhinolaryngology, School of Medicine, University of Fukui, Matsuoka Shimoizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japanb
Biomedical Imaging Research Center, University of Fukui, Matsuoka Shimoizuki, Eiheiji-cho, Yoshida-gun, Fukui 910-1193, Japanc
Diagnostic Imaging Group, Molecular Imaging Center, National Institute of Radiological Sciences, Anagawa 4-9-1, Chiba 263-8555, Japand
Division of Radiological Sciences, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Received 14 October 2008; received in revised form 26 December 2008; accepted 31 January 2009

Abstract
The purpose of this study is to reveal characteristics of 64Cu-labeled diacetyl-bis(N4-methylthiosemicarbazone) ([64Cu]Cu-ATSM) during
cell proliferation and hypoxia by autoradiography imaging and immunohistochemical staining.
Methods: The intratumoral distributions of [64Cu]Cu-ATSM and [18F]-2-fluoro-2-deoxy-D-gloucose ([18F]FDG) in mice implanted with
Lewis lung carcinoma (LLC1) tumor cells according to dual autoradiography were compared with the immunohistochemical staining patterns
of proliferating markers [Ki-67 and 5-bromo-2′-deoxyuridine (BrdU)] and a hypoxic marker (pimonidazole). A clonogenic assay was
performed using the cells of LLC1 tumor-implanted mice, and it was compared with the distribution of [64Cu]Cu-ATSM.
Results: [64Cu]Cu-ATSM mainly accumulated at the edge of tumors, whereas [18F]FDG was distributed inside the tumor and inside the[64Cu]Cu-ATSM accumulation. The number of Ki-67-positive cells/area tended to increase with [18F]FDG accumulation and decrease with
[64Cu]Cu-ATSM accumulation. On the other hand, the number of BrdU-positive cells/area was negatively correlated with [18F]FDG
accumulation and positively correlated with [64Cu]Cu-ATSM accumulation. High [64Cu]Cu-ATSM accumulation was found outside thehigh-[18F]FDG-accumulation and pimonidazole-positive regions. Colony formation ability was significantly higher in the tumor cells
obtained from high-[64Cu]Cu-ATSM-accumulation regions than the cells from the intermediate- and the low-accumulation regions.
Conclusion: [64Cu]Cu-ATSM accumulation regions in tumor cells indicate quiescent but clonogenic tumor cells under mild hypoxia. [64Cu]
Cu-ATSM could play an important role in planning appropriate tumor radiotherapy.
© 2009 Elsevier Inc. All rights reserved.
Keywords: [64Cu]Cu-ATSM; [18F]FDG; Proliferation; Hypoxia; Clonogenic assay

1. Introduction
Hypoxia is a key microenvironmental factor for tumor
development; not only does it stimulate angiogenesis andglycolysis for tumor expansion, but it also induces cell
cycle arrest and genetic instability with tumor progression[1]. In addition, hypoxic regions in solid tumors are
known to be resistant to radiotherapy as well as chemotherapy [2]. Thus, precise detection of hypoxic regions in⁎ Corresponding author. Tel.: +81 776 61 8431; fax: +81 776 61 8170.
E-mail addresses: sampo@u-fukui.ac.jp (M. Oh), yfuji@u-fukui.ac.jp(Y. Fujibayashi).
0969-8051/$ – see front matter © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.nucmedbio.2009.01.016

tumors is of importance to predict tumor malignancy andtherapeutic outcome.
Radio-copper-labeled Cu-diacetyl-bis(N4-methylthiosemicarbazone) (Cu-ATSM) has been developed as a positronemission tomography (PET) agent for hypoxia imaging[3–6] as well as an internal radiotherapy agent that allows
selective delivery of β-emitting Cu nuclides [7,8]. Clinicalstudy has indicated the usefulness of radio-copper-labeled
Cu-ATSM for predicting the prognosis of radiotherapy inseveral types of cancer [9,10]. A basic comparative study of[ 64 Cu]Cu-ATSM and immunohistochemical staining
revealed that high-[64Cu]Cu-ATSM regions demonstrate
fewer Ki-67-positive “proliferating” cells and lower vascularity, but a slight increase in apoptotic cells (although this420

M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

was less than 1%), when compared with low-[64Cu]CuATSM regions [11]. These findings are consistent with the
known characteristics of hypoxic tumor masses.
However, we also found that intratumor [18F]-2-fluoro-2deoxy-D-gloucose ([18 F]FDG) uptake, an indication of
glycolysis, was not positively correlated with “hypoxia” asshown by [64Cu]Cu-ATSM uptake [11,12]. In addition, theintratumor distribution of [64Cu]Cu-ATSM is reported to bedifferent from that of F-18-fluoromisonidazole ([18 F]FMISO), a traditional hypoxia marker [13,14]. Considering
these differing findings, radio-copper-labeled Cu-ATSM
might visualize different aspects of hypoxia than traditional
nitroimidazole compounds such as [18F]F-MISO.
Supply of oxygen as well as nutrition is limited to therange of 100 to 200 μm from the vessel, and outside thisrange, it should become necrotic. Positron emission
tomography with 3- to 5-mm resolution cannot visualize
such microenvironment; hence, the hypoxic region in the
PET image should be evaluated as a mixture or an average ofheterogenous phenotypes.
In the present study, we compared the intratumor
distribution of [64Cu]Cu-ATSM with [18F]FDG, a marker
of glycolysis that is known to be enhanced under hypoxic
conditions, macroscopically. Pimonidazole staining was also
performed as a “low-oxygen, tension-specific” probe, atmacroscopic as well as microscopic levels. The intratumor
distribution of the three “hypoxia”-seeking probes withdifferent aspects was compared with immunohistochemical
staining of Ki-67 and 5-bromo-2′-deoxyuridine (BrdU) to
elucidate the regional proliferation status, which is considered as an important feature of tumor cells. Regional
clonogenicity was also examined as another aspect of tumor
cells. Based on these results, possible interpretation of PET
images obtained with radiolabeled Cu-ATSM, FDG andnitroimidazole probes was discussed.
2. Materials and methods
2.1. Radiopharmaceutical synthesis
64

Cu was produced in a small biomedical cyclotron at
the Biomedical Imaging Research Center at the Universityof Fukui, Japan, according to a published method [12].
[64Cu]Cu-ATSM was synthesized by mixing 200 mM ofglycine buffer containing 64Cu and H2ATSM in dimethyl
sulfoxide (1:100 by mole ratio), as described previously[6]. The radiochemical purity of synthesized [64Cu]CuATSM was N99%, as evaluated by high-performance liquid
chromatography (LC-10ADVP; Shimadzu, Kyoto, Japan)
using a reversed-phase column (Cosmosil 5C18-AR,
4.6×50 mm+4.6×150 mm; Nacalai Tesque, Kyoto, Japan)[15]. [18 F]FDG was synthesized by the method of
Hamacher et al. [16] with an automated [18 F]FDG
synthesizing system (JFE, Tokyo, Japan). The specificactivity of [64Cu]Cu-ATSM was 56 GBq/μmol, and that of[18F]FDG was 20 to 50 GBq/μmol.

2.2. Animal model
Mice were treated in accordance with the animaltreatment guidelines of the University of Fukui throughout
the experiments. Male C57BL/6 mice (10 weeks old,weighing 20–25 g) were obtained from Japan SLC(Shizuoka, Japan). Approximately 107 Lewis lung carcinoma (LLC1) cells suspended in phosphate-buffered saline(PBS) were subcutaneously implanted into the right flankof mice.
2.3. Autoradiographic study
At 3 weeks after the implantation of tumor cells, eachmouse was injected intravenously with 92.5 MBq (2.5 mCi)of [18F]FDG, 463 kBq (12.5 μCi) of [64Cu]Cu-ATSM and
BrdU (10 μg/g of body weight; Sigma-Aldrich, St Louis,
MO, USA) or pimonidazole hydrochloride [1-([2-hydroxy-3piperidinyl]propyl)-2-nitroimidazole hydrochloride] (60 μg/
g of body weight, HP1-100, Hypoxyprobe-1 Kit for thedetection of tissue hypoxia; CHEMICON, Temecula, CA,
USA). Sixty minutes after the injection, the mice weresacrificed and the tumors were removed. The removed
tumors were immediately covered with optimal cutting
temperature compound and frozen in methanol cooled withdry ice. They were divided into two sections and frozen, andthe cutting surfaces were flattened with a cryostat (Cryocut
1800; Leica, Wetzlar, Germany) and subjected to dual-tracer
autoradiography [5]. [18F]FDG images were acquired over
3 min by exposing the frozen sections to an imaging plate(BAS-MP 2040S; Fuji Photo Films, Japan) in a freezer. The
imaging plate was scanned with a bioimaging analyzer (BAS1500, Fuji Photo Films). After waiting 40 h for 18F decay,[64Cu]Cu-ATSM images were acquired over 45 h underfrozen conditions, and the imaging plate was scanned. The
distributions of [18F]FDG and [64Cu]Cu-ATSM were visualized by Mac-BAS v2.52 software (Fuji Photo Films). The
contribution of 64Cu radioactivity to the FDG image (the first
autoradiography) was estimated to be around 1%, and thecontribution of 18F radioactivity to the Cu-ATSM image (thesecond exposure) was thought to be less than 0.1%. In eachtumor section, the most photostimulated luminescence region
was classified as 100%, and the background was defined as
0%. The 0% to 100% range was divided into four parts andcolored red (75–100%), orange (50–75%), green (25–50%)
and blue (0–25%), while the background was covered black.
The colored image was saved in true color TIFF format.
2.4. Immunohistochemical staining with Ki-67 and BrdU
The frozen blocks used for the double tracer autoradiography were thawed, fixed in 10% neutral buffered formalin
and embedded in paraffin. The sections used for the
immunohistochemical staining were taken from a region
50 μm from the surface exposed for autoradiography. After64
Cu decay, immunohistochemical staining was carried out
to detect proliferating cells, using 4-μm-thick serial paraffinM. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

sections. The proliferation markers used were Ki-67 and
BrdU incorporation into DNA.
Ki-67 is a nuclear protein that is expressed at all active
phases of the cell cycle (G1, S, G2 and mitosis) with its
highest expression being in the G2/M phase, but it is absent
from resting cells (G0) [16]. The proliferating cell fraction
can be determined immunohistochemically by using antibodies against Ki-67. The sections were deparaffinized andrehydrated, and then endogenous peroxidase was blocked
by 3% hydrogen peroxide. Antigen retrieval was carried out
by microwaving for 25 min in 10 mM of citrate buffer at
pH 6. Nonspecific stain-blocking reagent (X0909; Dako
Cytomation, Glostrup, Denmark) was applied for 20 min atroom temperature (RT). The sections were then incubatedovernight at 4°C with rat monoclonal anti-mouse Ki-67
antigen antibody (M7249, Dako Cytomation) in a 1:50
dilution with PBS. After washing with PBS, the sections
were incubated with rabbit anti-rat biotinylated secondary
antibody (E0468, Dako Cytomation) in a 1:200 dilution
with PBS for 30 min at RT before being incubated withstreptavidin conjugated to horseradish peroxidase (K0673,
Dako Cytomation) for 30 min at RT. Finally, the sections
were incubated with 3,3′-diaminobenzine tetrahydrochloride solution (DAB liquid system, Dako Cytomation) until
suitable staining developed, and then the nuclei wereslightly counterstained with hematoxylin.
BrdU is a halogenated pyrimidine analogue of thymidine,
which is incorporated into the DNA of proliferating cells in
the S-phase; thus, it tracks the fate of dividing cells and theirprogeny. BrdU can be detected directly with an anti-BrdU
monoclonal antibody [17,18]. It is generally used as aproliferating marker. After the sections were deparaffinized
and rehydrated, endogenous peroxidase was blocked by 3%hydrogen peroxide and the sections were incubated withprotease K (Wako, Japan) in 1:50,000 dilutions with PBS for
20 min at 37°C. After rinsing with PBS, the sections wereincubated with 2 N HCl for 15 min at 45°C and neutralizedwith 0.1 M borate buffer two times at 5 min each before
being rinsed with PBS containing 0.02% Tween-20, andnonspecific staining was blocked as above. The sections
were incubated overnight at 4°C with mouse monoclonal
antibody against BrdU (Roche Diagnostics, Switzerland) in
1:200 dilutions with PBS. After washing with PBS containing 0.05% Tween-20 (PBST-0.05%), the sections wereincubated with rabbit anti-mouse biotinylated secondary
antibody (Dako Envision+ Dual Link; Dako Cytomation,
Japan) for 30 min at RT. After washing with PBST-0.05%,
peroxidase color visualization was carried out with DAB (30mg dissolved in 150 ml of PBST-0.05% added to 10 μl of
30% H2O2 solution; Dojin, Kumamoto, Japan). Counterstaining was performed as described above.
2.5. Immunohistochemical staining with a hypoxic marker
Pimonidazole hydrochloride is a bioreductive chemicalprobe with an immunorecognizable side chain. The addition421

of the first electron during bioreductive activation isreversibly inhibited by oxygen, resulting in futile cycling
with a half-maximal pO2 of inhibition of about 3 mmHg,
with complete inhibition occurring at about 10 mmHg [19].
The sections were deparaffinized and rehydrated, and thenendogenous peroxidase was blocked by 3% hydrogen
peroxide. Nonspecific stain-blocking reagent was applied
for 15 min at RT. The sections were then incubated overnightat 4°C with hypoxyprobe (hypoxyprobe Mab-1, Hypoxyprobe-1 Kit for the Detection of Tissue Hypoxia, CHEMICON) in a 1:10 dilution with PBS. After washing with PBS,
the sections were incubated with rabbit anti-mouse biotinylated secondary antibody for 50 min at RT. After washingwith PBS, peroxidase color visualization was carried out
with DAB solution, and counterstaining was performed as
described above.
2.6. Autoradiography image analyses withproliferation markers
Whole images of the serial sections stained for Ki-67,
BrdU or pimonidazole were captured by a scanner (Epson
GT-8500) and saved in JPEG format. Composite images
were made using Adobe Photoshop to compare theautoradiographic images with those of the stained sections.
[64Cu]Cu-ATSM and [18F]FDG images were stacked inlayers above the images of stained sections and madetranslucent. A composite image was made for each tumor
mass, and five composite images were quantified. In eachcomposite image, three areas from the region of each color
for [64Cu]Cu-ATSM and [18F]FDG were analyzed. Digital
images of the area (0.31 mm2) were obtained at magnification ×200 using a microscope (Olympus BX50) mountedwith a CCD camera and connected to a Windows computer.
Positive cells were manually counted on the computer
monitor. The numbers of positively stained cells in the 15
areas (three areas from each section, one section from each of
five mice) were averaged for each color indication level of[64Cu]Cu-ATSM and [18F]FDG accumulation.
2.7. Clonogenic assay
Lewis lung carcinoma tumor-bearing mice prepared as
described above were intravenously injected with 3.7
MBq [64 Cu]Cu-ATSM. After 1 h, the mice weresacrificed and had their tumors removed, and two serial
slices 1-mm thick were cut from each tumor mass
immediately. From one slice, three to five blocks (2×2×1mm) were cut. The blocks were minced, treated with 0.1
mg/ml collagenase II (Sigma) for 20 min at 37°C,
disrupted by pipetting and filtered through a mesh toremove debris. The cells were recovered by centrifugation
and resuspended in culture medium. The recovered cells
were counted, and 500 cells were seeded in a 10-cm
culture plate in duplicate. After 7 days, formed colonies
were counted. The other slice was exposed to an imaging
plate, and accumulation of [64Cu]Cu-ATSM was analyzed

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M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

by MacBas as described above. The positions of theblocks were referred to the [64Cu]Cu-ATSM image, and[64Cu]Cu-ATSM accumulation of the regions from which
the blocks were cut out was determined and classified
into low (in the areas colored blue to green, 0–50%),
intermediate (mostly orange, 50–75%) and high (mostly
red, 75–100%). Out of 18 blocks in total from four mice,
five were classified as low, five as intermediate and eight
as high.
Statistical analysis was performed with the Wilcoxon
rank-sum test using Office Excel 2003 software (Microsoft).
Pb.05 was considered statistically significant.
3. Results
Fig. 1. Representative intratumoral distribution of [18F]FDG and [64Cu]CuATSM in the tumor mass of LLC1. The autoradiographic images of [18F]
FDG and [64Cu]ATSM are displayed in the same section. [64Cu]Cu-ATSM ismainly accumulated at the edge of the tumors, and no accumulation is seenin the center where the cells are necrotic. The highest uptake region of [64Cu]
Cu-ATSM was outside that of [18F]FDG.

3.1. Intratumor distribution of [18F]FDG and[64Cu]Cu-ATSM
To analyze the intratumor distributions of [64Cu]CuATSM and [18F]FDG, we performed dual autoradiography
with mouse-implanted LLC1 tumors. Representative images
are shown in Fig. 1. [64Cu]Cu-ATSM mainly accumulated at

Fig. 2. Immunohistochemical staining for Ki-67 (A and B) and BrdU (C and D) in LLC1 cells at ×200. The highest [18F]FDG regions are in Panels A and C,
while the [64Cu]ATSM regions are in Panels B and D. Positive nuclear staining of Ki-67 in tumor cells was abundantly observed in the highest [18F]FDG region,but it was hardly observed in that of [64Cu]Cu-ATSM. BrdU-positive cells were hardly observed in the highest [18F]FDG region, but they were abundantly
observed in that of [64Cu]Cu-ATSM.

M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

the edge of the tumors, and no accumulation was seen in thecenter where the cells were necrotic. On the other hand, thehighest uptake region of [18F]FDG was seen inside that of[64Cu]Cu-ATSM. The most intense regions of [18F]FDG and[64Cu]Cu-ATSM staining, which were colored red, were
distributed differently in all sections studied. These results
were consistent with our previous report [11].
3.2. Autoradiography image analysis with Ki-67
To evaluate cell proliferation in each section, we performed
Ki-67 and BrdU immunohistochemistry. Positive nuclear
staining of Ki-67 in tumor cells was abundantly observed in
the highest [18F]FDG region, but it was hardly observed inthat of [64Cu]Cu-ATSM (Fig. 2A and B). There was atendency for the number of Ki-67-positive cells to increase
with [18F]FDG uptake (Fig. 3A) and to decrease with [64Cu]
Cu-ATSM uptake (Fig. 3B) as in our previous report [11].

423

3.3. Autoradiography image analysis with BrdU
BrdU-positive cells were hardly observed in the mostintensely fluorescent [18F]FDG regions, but they wereabundantly observed in those of [64Cu]Cu-ATSM, contrary
to the distribution of Ki-67-positive cells (Fig. 2C and D).
There was a negative correlation between the number of
BrdU-positive cells and [18F]FDG uptake (Fig. 3C), andthere was a positive correlation between the number of
BrdU-positive cells and [64Cu]Cu-ATSM uptake (Fig. 3D).
3.4. Autoradiography image analysis with pimonidazole
In LLC1 tumor, [18F]FDG was distributed inside the tumor,and [64Cu]Cu-ATSM was outside the distribution of [18F]
FDG, same as above. Fig. 4 shows an image of immunochemical staining with pimonidazole and an autoradiographic
image of [18F]FDG and [64Cu]Cu-ATSM in LLC1 tumor. [18F]
FDG accumulation is distributed inside the tumor, and [64Cu]

Fig. 3. Correlation between the number of Ki-67-positive (A and B) or BrdU-positive (C and D) cells and the accumulation of [18F]FDG (A and C) or [64Cu]ATSM(B and D). The number of Ki-67-positive cells tended to increase with [18F]FDG accumulation and decrease with [64Cu]Cu-ATSM accumulation. On the other
hand, the number of BrdU-positive cells was negatively correlated with [18F]FDG accumulation and positively correlated with [64Cu]Cu-ATSM accumulation.

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M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

Fig. 4. An immunohistochemical staining image of pimonidazole and an autoradiographic image of [18F]FDG and [64Cu]Cu-ATSM at 1 h after injection into
LLC1 cells. The pimonidazole staining image and the (A) autoradiographic images of [18F]FDG (B) and [64Cu]Cu-ATSM (C) were presented on the same
slice. [18F]FDG accumulation was distributed inside the tumor, and [64Cu]Cu-ATSM accumulation was distributed outside the distribution of [18F]FDG. The
hypoxic regions shown by staining pimonidazole hydrochloride are inside the tumor and generally overlap with the distribution of [18F]FDG but not [64Cu]
Cu-ATSM.

Cu-ATSM accumulation is distributed outside the distribution of [18F]FDG. Pimonidazole hydrochloride staining of
hypoxic regions is shown inside the tumors and generally
lapped over the distribution of [18F]FDG, but not [64Cu]CuATSM at 1 h postinjection of the radiotracers.
3.5. Clonogenic assay
Proliferation potential of the cells in LLC1 tumor masses
was assessed by a colony formation assay. Table 1 shows thenumber of colonies formed from the cells recovered from theregions of high, intermediate and low accumulation of [64Cu]
Cu-ATSM. The number was significantly higher in the high[64Cu]Cu-ATSM-accumulation region than in the intermedi-

ate- and the low-accumulation regions (Pb.05), indicatingthat the region of high [64Cu]Cu-ATSM accumulation had ahigher proliferation potential than the other regions.
Table 1
Colony formation ability of LLC1 tumor cells from various regions[64Cu]Cu-ATSM
accumulation

No. of regions

No. of colonies formed/100
cells (average±S.D.)

High
MediumLow

85520.1±3.2a
14.5±4.0
12.9±5.1

a
The number of colonies in the high-[64Cu]Cu-ATSM-accumulation
region was significantly higher than that in the intermediate- and the lowaccumulation regions (Pb.05).

M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

4. Discussion
Copper-ATSM is a stable Cu(II) complex with highmembrane permeability and is able to pass through the cell
membrane between the blood and tissues instantly. Under
abnormally high-NADH conditions such as hypoxia,
however, Cu(II) in the Cu(II)-ATSM complex can be easily
reduced to Cu(I) by a mitochondrial electron transport
enzyme (NADH dehydrogenase) in an NADH-dependent
manner. Hypoxia-selective reduction occurs in tumor cells
also [4], but reduction in tumors is mediated by microsome
fraction, not by mitochondria [20]. Microsomal reductionof Cu-ATSM is regulated by NADH-cytochrome b5
reductase and/or NADPH-cytochrome P450 reductase in
an NADH/NADPH-dependent manner, and reduced Cu(I)is instantly dissociated from ATSM and retained within thecells. Thus, the key to Cu-ATSM retention is notconsidered to be oxygen tension itself, but rather a high
NADH/HADPH concentration mostly, which occurs as aresult of hypoxia.
Also, in the case of nitroimidazole compounds such as
pimonidazole, reductive activation is ubiquitously performed by redox enzymes in an NADH/NADPH-dependent
manner [21]. However, the produced nitro anion radical canbe easily reoxidized under normoxic conditions [22,23]. As aresult, retention of nitroimidazole is regulated by oxygen
tension but is independent of NADH/NADPH redox state.
From these points of consideration, Cu-ATSM and pimonidazole show completely different aspects of hypoxic tumor
cells, namely, the highly reduced status and low oxygen
tension within the cells, respectively.
At the macroscopic level shown in Fig. 4, pimonidazole
staining showed an almost identical pattern to those of[18F]FDG at 1 h postinjection. The necrotic region (nostaining, no [18F]FDG uptake) was surrounded by ahypoxic region (pimonidazole positive, high [18F]FDG
uptake) and then by a normoxic region (pimonidazole
negative, moderate [18F]FDG uptake). The key point hereis that the distances between necrosis–hypoxia–normoxia
were more than 1 to 3 mm. However, the pimonidazolepositive region was not homogenous but a network ofblood vessels surrounded by proliferating tumor cells,
hypoxic tumor cells and necrosis. This patchy pattern isrepeated within 100 to 200 μm, known as the distance thatis penetrable by oxygen. From this finding, the “hypoxic”
region detected by PET using radiolabeled nitroimidazole
compounds and [18F]FDG is not a homogenously hypoxic
region but rather is a mixture of rapidly proliferatingtumor cells, hypoxic nonproliferating cells and necrosis.
High accumulation of radiolabeled nitroimidazole compounds is considered to only occur in hypoxic nonproliferating cells. [18F]FDG accumulation is considered to beenhanced in both regions of normoxic proliferating cells
and hypoxic nonproliferating cells, because of highdemand of energy for proliferation and anaerobic glycolysis, respectively. From these findings, it is indicated that425

“hypoxia” imaging with PET using [ F]FDG or [ F]FMISO does not simply visualize hypoxic cells, but amixture of various types of cells in tumor masses,
including cells under low oxygen tension. In addition,high [64Cu]Cu-ATSM accumulation at 1 h postinjection
was found outside the high-[18F]FDG/pimonidazole-positive region. This result corresponded with our previous
report using [64Cu]Cu-ATSM and [18F]FDG [11]. Considering the high [64 Cu]Cu-ATSM uptake, lack ofpimonidazole staining and the retention mechanisms ofboth agents, this region is thought to be in an extremely
reductive condition, but not severe hypoxia.
In Fig. 2, BrdU-positive cells were hardly observed in thehigh-[18F]FDG regions but were observed in the high-[64Cu]
Cu-ATSM regions, indicating that cells with a high [64Cu]
Cu-ATSM uptake have the ability to synthesize DNA but notduring the proliferation process. BrdU is used for thedetection of cell proliferation by means of DNA synthesis,
especially the detection of S-phase cells. Ki-67 is also
reported as a marker of proliferation, and it expresses allphases of proliferation except for G0. However, theexpression of Ki-67 varies in intensity throughout the cell
cycle, and this has raised concern that it could lead to amisclassification of cycling cells as the resting ones [24]. The
levels of Ki-67 are low during the G1- and early S-phase andprogressively increase to reach their maximum duringmitosis [24]. van Dierendonck et al. [25] noted that some
BrdU-positive cells are Ki-67 negative, although all BrdUpositive cells are thought to be Ki-67 positive. This isprobably due to methodological bias [26]. In addition, mild
hypoxia (ca. 1%) shows transient increases in the number of
BrdU-positive cells [27], and some cancer cell lines are able
to continue BrdU incorporation for as long as 24 to 48 hunder these conditions [28].
Pooling these findings, it can be suggested that thecells in high-[64Cu]Cu-ATSM regions were quiescent but
continued DNA synthesis and that this status was inducedby mild hypoxia. Extracellular conditions were considered
to be rather similar within the wide range of regions
because of the quiescence of the cells. This type of cells
remained sensitive to progression factors; the cells
obtained from a high-[64Cu]Cu-ATSM region showed ahigh clonogenic ability under optimal growth conditions(Table 1). Therefore, the [64Cu]Cu-ATSM accumulation
regions were considered to be clonogenic tumor cells.
These tumor cells should be attacked with [64Cu]CuATSM internal radiotherapy and/or X-ray external radiotherapy, such as intensity-modulated radiation therapy. On
the other hand, in high-[18F]FDG and pimonidazolepositive regions, complex cell populations existed; somewere actively proliferating, but the large oxygen gradientfrom blood vessels to areas of necrosis might induce awide variety of proliferation statuses/abilities within thecells. As a result, the overall clonogenic ability of high[18F]FDG areas was lower than that of high-[64Cu]CuATSM regions.
18

18

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M. Oh et al. / Nuclear Medicine and Biology 36 (2009) 419–426

5. Conclusion
We have demonstrated that regions of accumulation of[64Cu]Cu-ATSM indicate quiescence in tumor cells andclonogenic tumor cells under mild hypoxia in an LLC1bearing animal model. [64Cu]Cu-ATSM would be a tool toprovide information on disease prognosis and radiotherapy
planning, though further basic as well as clinical studies
would be needed to clarify its precise usefulness.
Acknowledgments
The authors thank Shingo Kasamatsu, Satonao Nakakoji and the rest of the staff of the Biomedical Imaging
Research Center and Nobuo Takimoto of the Departmentof Pathology at the University of Fukui, Japan. This
study was partly funded by a Grant-in-Aid in Scientific
Research from the Japan Society for the Promotion of
Science (20791185) and the 21st Century COE Program(Medical Science).
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